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Cardiovascular Research 2003 59(4):883-892; doi:10.1016/S0008-6363(03)00517-0
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Copyright © 2003, European Society of Cardiology

Tamoxifen and ICI 182,780 negatively influenced cardiac cell growth via an estrogen receptor-independent mechanism

Isabelle Merciera,b, Sylvie Maderc and Angelino Calderonea,b,*

aDepartment of Physiology, University of Montreal, Montreal, Quebec, Canada
bMontreal Heart Institute, Research Center, 5000 Belanger Street East, Montreal, Quebec, Canada H1T 1C8
cDepartment of Biochemistry, University of Montreal, Montreal, Quebec, Canada

calderon{at}icm.umontreal.ca

* Corresponding author. Tel.: +1-514-376-3330x3710; fax: +1-514-376-1355.

Received 13 March 2003; revised 5 June 2003; accepted 7 July 2003


   Abstract
Top
 Abstract
1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 
Background: Recent studies have demonstrated that selective estrogen receptor modulators (SERMs) reduced identifiable risk factors implicated in cardiovascular disease. Despite this observation, the direct effect of SERMs on cardiac cell growth remains unexplored. Methods: Neonatal rat cardiac myocytes (CM) and fibroblasts (CF) were exposed to either the partial estrogen receptor agonist/antagonist 4-OH tamoxifen (e.g., SERM) or the pure estrogen receptor antagonist ICI 182,780 and the effect on DNA synthesis, cell cycle protein expression and extracellular signal-regulated kinase (ERK1/2) phosphorylation were assessed. Results: The treatment of CM and CF with either 4-OH tamoxifen or ICI 182,780 decreased DNA synthesis in the absence of apoptosis via an estrogen receptor-independent pathway. In CM and CF, 4-OH tamoxifen and ICI 182,780 treatment reduced proliferating cell nuclear antigen protein expression and concomitantly increased p27Kip1. 4-OH Tamoxifen and ICI 182,780 treatment increased ERK1/2 phosphorylation in CM and CF, and ERK1/2 kinase (MEK)-dependent inhibition of ERK1/2 activation attenuated ICI 182,780-mediated suppression of DNA synthesis. Conclusion: These data are the first to describe cardiac cells as novel targets of SERMs and ICI 182,780, and highlight the role of the ERK1/2 pathway in the suppression of DNA synthesis.

KEYWORDS Cell culture/isolation; Signal transduction; Protein kinases; Receptors; Remodeling


   1. Introduction
Top
 Abstract
 1. Introduction
2. Methods
 3. Results
 4. Discussion
 References
 
It has been established that pre-menopausal women have a lower risk of cardiovascular disease, as compared to age-matched men [1]. Following the onset of menopause, the incidence of cardiovascular disease in women increased to levels comparable to that observed in men [2]. Consequently, it has been proposed that ovarian hormones represent an intrinsic cardio-protective mechanism, and thus hormonal replacement therapy (HRT) in post-menopausal women has received considerable attention [3,4]. However, this latter concept was recently challenged by the Heart and Estrogen/Progestin Replacement Study (HERS) as HRT failed to demonstrate an overall cardio-protective action, and further increased the incidence of thromboembolism in post-menopausal women [5]. Likewise, in the Women’s Health Initiative study, increased risk of pulmonary embolism, coronary artery disease, and invasive breast cancer were documented in post-menopausal women receiving HRT [6].

Tamoxifen and raloxifene are representative of a family of molecules designated selective estrogen receptor modulators (SERMs). The pharmacological profile of these compounds is tissue selective, acting either as a partial estrogen receptor agonist or antagonist [7]. Tamoxifen is an effective antagonist in the treatment of estrogen-dependent breast cancer, whereas in bone, both tamoxifen, and raloxifene mimic the beneficial action of estrogen [7]. In addition, SERMs reduced identifiable cardiovascular risk factors, including serum cholesterol, LDL, and fibrinogen B [7–10]. Surprisingly, and in contrast to HRT, the incidence of coronary artery disease and fatal MI in breast cancer patients receiving tamoxifen were reduced, as compared to placebo [11,12]. In the MORE (Multiple Outcomes of Raloxifene Evaluation) study, raloxifene treatment significantly reduced the incidence of cardiovascular events in a subset of women characterized with increased cardiovascular risk [13]. Thus, SERM therapy may be efficacious in ameliorating the risk of cardiovascular disease-related events in post-menopausal women.

The direct effects of SERMs on cardiac cells remain unexplored, despite the observation that myocytes and fibroblasts have been identified as targets of ovarian hormones [14,15]. Interestingly, a novel receptor that binds exclusively to SERMs has been identified, and may contribute in part to the reported antiproliferative action of tamoxifen documented in estrogen receptor-negative cell lines [16–19]. Moreover, in the canine ventricle, tamoxifen inhibited calcium uptake by the sarcoplasmic reticulum via an estrogen receptor-independent mechanism [20]. Surprisingly, in an estrogen receptor-negative ovarian carcinoma cell line, the pure steroidal estrogenic receptor antagonist ICI 182,780, which has no known estrogenic properties, mimicked the antiproliferative and apoptotic actions of tamoxifen [19]. Lastly, ICI 182,780 and SERMs inhibited angiogenesis and this response was not altered in the presence of excess estrogen [21]. Thus, the present study examined whether tamoxifen and raloxifene influenced cardiac cell growth via either an estrogen receptor-dependent or -independent mechanism. Lastly, if an estrogen receptor-independent mechanism was implicated, a second series of experiments were performed to assess whether the pure estrogen receptor antagonist ICI 182,780 mimicked the action of SERMs on cardiac cells.


   2. Methods
Top
 Abstract
 1. Introduction
 2. Methods
3. Results
 4. Discussion
 References
 
2.1 Preparation of cultured neonatal rat ventricular myocytes and fibroblasts
Cardiac myocytes and fibroblasts were isolated from 1- to 3-day-old Sprague–Dawley rat pups (Charles River Canada) as previously described [15]. Experiments were performed in accordance with the principles of the Canadian Council on Animal Care and approved by the Animal Ethics Committee of the Montreal Heart Institute. Cardiac cells were cultured in phenol red-free media DMEM containing 7% heat-inactivated and treated-FBS (treated with 1% activated carbon Norit SA3, and Dextran T70 to remove endogenous steroids).

2.2 DNA synthesis experiments
Cardiac myocytes (200–400 cells/mm2) and fibroblasts (Passage 1; 100–200 cells/mm2) were plated in phenol red-free DMEM-containing 7% heat-inactivated and treated-FBS for 24 h, subsequently washed, and maintained in phenol red-free DMEM-containing insulin (5 µg/ml), transferring (5 µg/ml), and selenium (5 ng/ml) (ITS) for 24–48 h prior to the experimental protocol. Cardiac cells were treated for a period of 24 h with 17β-estradiol (Sigma, MI), 4-OH tamoxifen (Sigma, MI), raloxifene (provided to Dr. Mader by Eli Lilly) or ICI 182,780 (provided to Dr. Mader by AstraZeneca). ICI 182,780, raloxifene, and 4-OH tamoxifen were dissolved in DMSO at a stock concentration of 0.01 M. The final concentration of DMSO at the highest dose of either ICI 182,780, raloxifene, or 4-OH tamoxifen was 1;10 000. In a separate series of experiments, the MEK inhibitor PD098059 (20 µM; Biomol, PA) or 17β-estradiol (1 µM) was added 30 min prior to the administration of ICI 182,780, and/or 4-OH tamoxifen. DNA synthesis was assessed by the addition of 1 µCi/ml of [3H]thymidine (ICN Biomedicals; Costa Mesa, CA) for a period of 4–6 h prior to the end of the treatment protocol. Protein synthesis was assessed by the addition of 2 µCi/ml of [3H]leucine (ICN Biomedicals) for a period of 24 h. Cells were washed twice with PBS (4°C), and cold 5% TCA was added for 30 min to precipitate either DNA or protein. The precipitates were washed twice with cold water and re-suspended in 0.4 M NaOH. Aliquots were counted in a scintillation counter.

2.3 TUNEL assay
Cardiac cells were plated as described above, and subsequently fixed for 10 min in paraformaldehyde (4%) and permeabilized with saponin solution (0.075% saponin/1 µM EGTA) for 15 min at room temperature and treated with RNase (5 mg/ml) to digest all non-DNA material. TUNEL assay was performed according to the manufacturer’s instructions (Boehringer-Mannheim, Germany). The reaction was terminated following the addition of a stop solution (500 mg of powdered skim milk in 20 ml of sodium chloride (0.15 M)/sodium citrate (0.1 M) (pH 7; SSC 4x). The nuclei were stained following the addition of 4 µl of extrAvidin-FITC per 200 µl of staining solution (500 mg of dry skim milk, 0.1% Triton X-100 in 10 ml of SSC 4x) for 30 min at room temperature. Nuclei were co-stained with 0.01 µM of propidium iodide for 15 min at room temperature. The coverslips were mounted on Dabco medium (0.2 µM), and the cells were observed with a x40 oil objective mounted on a Zeiss Axiovert 100 M confocal microscope.

2.4 Western blot analysis of estrogen receptor-{alpha}, -β, p27Kip1, PCNA, and ERK1/2 phosphorylation
The subcellular localization of the estrogen receptor-{alpha}, and -β subtypes was performed on cytosolic and particulate fractions, as previously described [15]. Briefly, cardiac myocytes (200–400 cells/mm2) and fibroblasts (Passage 1; 100–200 cells/mm2) plated in phenol red-free DMEM-containing 7% heat-inactivated and treated-FBS for 24–48 h, subsequently washed, and maintained in phenol red-free DMEM-containing ITS for 24–48 h. Cardiac cells were subsequently washed twice with PBS (pH 7.4), and re-suspended in 1 ml of a buffer containing 20 mM Hepes (pH 7.5), 20 mM β-glycerophosphate, 20 mM NaF, 0.2 mM Na3VO4, 5 mM EDTA, 5 mM EGTA, 0.5 mM PMSF, 25 µg/ml leupeptin, and 5 mM DTT (Buffer A). A fraction of the homogenate was treated with 2% Triton X-100 to obtain the whole cell extract and the remaining of the homogenate was centrifuged for 30 min at 100 000xg (4°C) to yield supernatant (cytosolic) and pellet fractions. The pellet was re-suspended in Buffer A plus 1% (v/v) Triton X-100, and centrifuged as before to yield a supernatant containing the particulate fraction. With regard to cell cycle protein expression and ERK1/2 phosphorylation, cardiac cells were plated at the same density as described above, kept in phenol red-free DMEM-containing 7% heat-inactivated and treated-FBS for 24–48 h, subsequently washed, and maintained in phenol red-free DMEM-containing ITS for 24–48 h. Following the experimental protocol, cardiac cells were lysed in a buffer containing 10 mM Tris (pH 7.5), 150 mM NaCl, 1 mM EDTA, 1 mM EGTA, 50 mM NaF, 0.5 mM phenylmethylsulfonyl fluoride, 1 mM sodium vanadate, 1% Triton X-100, 0.5% Nonidet P-40, and 1 µg/ml of leupeptin and aprotinin. The homogenate was centrifuged for 5 min, and the supernatant was frozen and stored at –80°C. The BioRad assay was used to determine protein content. Three hundred µg of cell lysate were subjected to SDS–polyacrylamide gel (10%) electrophoresis to assess estrogen receptor expression, whereas 100–200 µg of cell lysate were used to determine cell cycle protein expression and ERK1/2 phosphorylation. Following electrophoresis, protein was transferred to Hybond-C membrane (Amersham Canada), and equal loading of the samples was confirmed with Ponceau S staining. The membrane was pre-incubated in 10 mM Tris, pH 7.4, 150 mM NaCl and 0.1% Tween (v/v) (TBS-T buffer) containing 3% skim milk for 1 h at room temperature, and subsequently incubated overnight at 4°C with either 1 µg/ml of a rabbit-polyclonal antibody directed against estrogen receptor-{alpha} or -β subtypes (Santa Cruz Biotechnology, CA). p27Kip1 and PCNA protein expression were detected with 1 µg/ml of a goat-polyclonal (Santa Cruz Biotechnology) or a rabbit polyclonal antibody (Santa Cruz Biotechnology), respectively. The activated form of ERK1/2 was detected with a rabbit polyclonal antibody (1:1000) recognizing the phosphorylated residues Thr202/Tyr204 (Cell Signaling, MA). In a separate series of experiments, the MEK inhibitor PD098059 (20 µM; Biomol, PA) was added 30 min prior to the administration of ICI 182,780 or 4-OH tamoxifen, and the subsequent effect on ERK1/2 phosphorylation examined. Following incubation, the membranes were washed three times with TBS-T containing 3% skim milk, and subsequently incubated for 1 h at room temperature with the appropriate secondary antibody (1:10 000) conjugated to horseradish peroxidase. Following incubation, the membranes were washed three times with TBS-T and the bands were detected by ECL detection kit. Films were scanned with a laser densitometer utilizing the program Quantity One (BioRad Laboratories, Canada).

2.5 Statistics
Data are represented as the mean±S.E.M, and (n) represents an independent preparation of cardiac myocytes and fibroblasts isolated from a litter of neonatal rat pups (eight to 12 pups per litter). Statistical analysis of dose–response curves on DNA synthesis was assessed by an ANOVA. Cell cycle protein expression, and ERK1/2 phosphorylation were evaluated by a Student’s unpaired t-test. A value of P<0.05 was considered as statistically significant.


   3. Results
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
4. Discussion
 References
 
3.1 SERMs and ICI 182,780 decreased DNA synthesis in cardiac cells without inducing apoptosis
In neonatal rat cardiac fibroblasts, the estrogen receptor-{alpha} subtype was detected in the cytosol and localized on the plasma membrane, whereas the estrogen receptor-β subtype was detected exclusively in the cytosol [15]. In neonatal rat cardiac myocytes, an antibody directed against the estrogen receptor-{alpha} detected an immunoreactive doublet at ~68 kDa in both the cytosolic and particulate fractions, albeit the level of expression was quantitatively greater in the cytosolic fraction (Fig. 1; n=2). Akin to cardiac fibroblasts, the estrogen receptor-β subtype was detected exclusively in the cytosolic fraction of neonatal rat cardiac myocytes (data not shown). The 24-h exposure of cardiac myocytes to 17β-estradiol did not influence either [3H]leucine (data not shown) or [3H]thymidine uptake (Fig. 1; n=4). By contrast, the 24-h exposure of cardiac myocytes to either 4-OH tamoxifen (n=5) or raloxifene (n=5) decreased DNA synthesis in a dose-dependent manner (Fig. 1), but did not influence protein synthesis (data not shown). The pure estrogen receptor antagonist ICI 182,780 (n=5) mimicked the SERM-dependent suppression of DNA synthesis (Fig. 1). These latter observations support the premise that SERM and ICI 182,780 mediated decrease of DNA synthesis in cardiac myocytes proceeded via an estrogen receptor-independent mechanism. To further validate this assumption, 17β-estradiol (1 µM) was employed as an antagonist and cardiac myocytes were pretreated for 30 min prior to the addition of either 4-OH tamoxifen (500 nM) or ICI 182,780 (100 nM). The 4-OH tamoxifen and ICI 182,780 mediated decrease of DNA synthesis in cardiac myocytes was quantitatively similar in the absence or presence of 17β-estradiol (Fig. 2).


Figure 1
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  		Fig. 1 Subcellular localization of estrogen receptor subtypes and the effect of SERMs and ICI 182,780 on DNA synthesis. (A) An immunoreactive doublet at ~68 kDa was detected in both the cytosolic (CYTO) and particulate (PART) fractions of cardiac myocytes, and corresponds to the estrogen receptor-{alpha} subtype (ER-{alpha}; n=2). WCE represents whole cell extract. (B) The treatment of cardiac myocytes with 17β-estradiol (open circle; n=5) had no effect on DNA synthesis. 4-OH Tamoxifen (solid square; n=5), raloxifene (open square; n=5), and ICI 182,780 (solid circle; n=5) treatment caused a significant dose-dependent decrease of [3H]thymidine uptake. (C) A decrease of [3H]thymidine uptake in neonatal rat cardiac fibroblasts was observed in response to 17β-estradiol (open circle) at a dose of 1 µM [15]. 4-OH Tamoxifen (solid square; n=5), raloxifene (open square; n=4), and ICI 182,780 (solid circle; n=5) treatment caused a significant dose-dependent decrease of [3H]thymidine uptake. *Denotes P<0.01 (ANOVA).

 

Figure 2
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  		Fig. 2 4-OH tamoxifen and ICI 182,780 inhibited DNA synthesis via an estrogen-receptor-independent mechanism. (A) The 24-h exposure of cardiac myocytes with 1 µM 17β-estradiol (E; n=5) had a minimal effect on [3H]thymidine uptake. 17β-estradiol (E; 1 µM) pretreatment with did not inhibit either 4-OH-tamoxifen (T; 500 nM; n=5) or ICI 182,780 (I; 100 nM; n=5) mediated suppression of DNA synthesis. (B) In cardiac fibroblasts, a 24-h exposure to 1 µM 17β-estradiol (E; n=3) decreased [3H]thymidine uptake. However, 17β-estradiol (E; 1 µM) pretreatment did not inhibit either 4-OH-tamoxifen (T; 500 nM; n=3) or ICI 182,780 (I; 100 nM; n=3) mediated suppression of DNA synthesis. The data are expressed as either percent change versus basal or 17β-estradiol.

 
In cardiac fibroblasts, we have previously demonstrated that the 24-h exposure to 17β-estradiol modestly decreased DNA synthesis at the maximal dose of 1 µM (Fig. 1) [15]. By contrast, the 24-h exposure to 4-OH tamoxifen (n=5), raloxifene (n=4), or ICI 182,780 (n=5) dose-dependently attenuated DNA synthesis, and the maximal response was greater than 17β-estradiol (Fig. 1). The pretreatment of cardiac fibroblasts with 17β-estradiol (1 µM) did not antagonize either 4-OH tamoxifen (500 nM) or ICI 182,780 (100 nM) mediated decrease of DNA synthesis (Fig. 2).

DNA fragmentation as assessed by the TUNEL assay, was not detected in cardiac fibroblasts following a 24-h exposure to either 4-OH tamoxifen (1 µM; n=2) or ICI 182,780 (1 µM; n=2) (Fig. 3). By contrast, a 30-min exposure to ultraviolet light (254 nm) resulted in a robust staining of the nuclei, indicative of DNA fragmentation (Fig. 3). In cardiac myocytes (data not shown), neither 4-OH tamoxifen (1 µM; n=2) nor ICI 182,780 (1 µM; n=2) treatment (24 h) induced DNA fragmentation.


Figure 3
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  		Fig. 3 Absence of apoptosis following 4-OH tamoxifen and ICI 182,780 treatment of cardiac fibroblasts. In untreated cardiac fibroblasts (Basal), apoptosis was not evident, as assessed by the TUNEL assay (TdT; n=2). Nuclear staining was observed with propidium iodide (PI) treatment (n=2). A 24-h treatment of cardiac fibroblasts with either 4-OH tamoxifen (TAM; 1 µM; n=2) or ICI 182,780 (ICI; 1 µM; n=2) did not induce DNA fragmentation (TdT). By contrast, a robust nuclear signal was detected in UV-treated cardiac fibroblasts (30 min), indicative of apoptosis (n=2). An overlap of the TdT and propidium iodide fluorescence signal was observed in the nucleus of UV-treated fibroblasts, as reflected by the emergence of a yellow fluorescence (TdT+PI).

 
3.2 Cell cycle protein regulation
The progression of the cell cycle from G1 to S phase (DNA synthesis) is regulated in part via the reciprocal regulation of the cyclin-dependent kinase inhibitor p27Kip1 and the DNA polymerase accessory protein proliferating cell nuclear antigen (PCNA). The 4-OH tamoxifen (1 µM) mediated decrease of DNA synthesis in cardiac fibroblasts was associated with increased p27Kip1 protein expression (110±31%; n=4; P<0.05 versus basal), and a concomitant decrease of PCNA protein level (32±12% {downarrow} versus basal; n=6; P<0.05) at 4 h, and was maintained at 24 h (p27Kip1, 113±44%; PCNA, 55±14% {downarrow}; n=4–6; P<0.05 versus basal) (Fig. 4). Likewise, ICI 182,780 (1 µM) treatment increased p27Kip1 (78±26%; n=3; P<0.05 versus basal) and concomitantly decreased PCNA (66±19% {downarrow}; n=5; P<0.05 versus basal) protein expression at 4 h, and was maintained at 24 h (p27Kip1, 73±25%; PCNA, 68±8% {downarrow}; n=3–5; P<0.05 versus basal) (Fig. 4).


Figure 4
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  		Fig. 4 Cell cycle protein regulation in cardiac fibroblasts. (A) 4-OH tamoxifen (TAM; 1 µM) and (B) ICI 182,780 (ICI; 1 µM) treatment of cardiac fibroblasts increased p27Kip1 and concomitantly decreased PCNA protein expression.

 
In cardiac myocytes, 4-OH tamoxifen (1 µM) treatment caused a modest non-significant increase of p27Kip1 protein expression at 4 h (64±36%; n=4; P=0.08), whereas a significant effect was observed at 24 h (50±21%; n=4; P<0.05 versus basal). By contrast, 4-OH tamoxifen significantly reduced PCNA protein at 4 (47±20% {downarrow}; n=4; P<0.05 versus basal) and 24 h (48±13% {downarrow}; n=5; P<0.05 versus basal). Likewise, ICI 182,780 (1 µM) treatment increased p27Kip1 (132±38%; n=5; P<0.05 versus basal) and concomitantly decreased PCNA (40±12% {downarrow}; n=5; P<0.05 versus basal) protein expression at 4 h. Following a 24-h exposure to ICI 182, 780, p27Kip1 protein levels remained elevated (138±44%; n=4; P<0.05 versus basal), whereas a modest non-significant decrease of PCNA protein expression was observed (30±15% {downarrow}; n=4; P=0.06).

3.3 Extracellular signal-regulated kinase (ERK) activity and subsequent role in DNA synthesis
In cardiac myocytes, the antihypertrophic action of the atrial natriuretic peptide required ERK activation [22]. In this regard, the contribution of ERK1/2 in 4-OH tamoxifen and ICI 182,780 mediated inhibition of DNA synthesis in cardiac cells was examined. ERK1/2 phosphorylation was increased at 5 min following the exposure of cardiac myocytes to either 1 µM 4-OH tamoxifen (ERK1=450±70%; ERK2=625±39%; n=5; P<0.01 versus basal) or ICI 182,780 (ERK1=356±58%; ERK2=506±30%; n=5; P<0.01 versus basal), and remained elevated at 30 min (Fig. 5). Likewise, ERK1/2 phosphorylation was increased in cardiac fibroblasts following a 5-min exposure to either 1 µM 4-OH tamoxifen (ERK1=360±39%; ERK2=266±34%; n=5; P<0.01 versus basal) or ICI 182,780 (ERK1=326±49%; ERK2=245±39%; n=5; P<0.01 versus basal) and remained elevated at 30 min (Fig. 5).


Figure 5
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  		Fig. 5 ERK1/2 phosphorylation in cardiac cells. The acute exposure of cardiac myocytes (A) or fibroblasts (C) to either 4-OH tamoxifen (TAM; 1 µM) or ICI 182,780 (ICI; 1 µM) rapidly increased ERK1 (p44) and ERK2 (p42) phosphorylation. The pretreatment of cardiac myocytes (B) or fibroblasts (D) with the MEK inhibitor PD098059 (PD; 20 µM) abrogated ERK1/2 phosphorylation by either 4-OH tamoxifen or ICI 182,780. Lastly, the acute exposure of cardiac myocytes (E) or fibroblasts (F) to 17β-estradiol (17β-E;1 µM) rapidly increased ERK1 (p44) and ERK2 (p42) phosphorylation.

 
The pretreatment of cardiac myocytes with the pharmacological agent PD098059 (20 µM), an inhibitor of the serine/threonine kinase MEK (upstream activator of ERK) abrogated 4-OH tamoxifen (91±5%{downarrow}; n=3) and ICI 182,780 (88±7%{downarrow}; n=3) stimulated ERK1/2 phosphorylation (Fig. 5). The 24-h exposure of cardiac myocytes to PD098059 (20 µM) had no effect on basal [3H]thymidine uptake (5±7% versus basal; n=4). However, the pretreatment (30 min) of cardiac myocytes with PD098059 (20 µM) abrogated the subsequent decrease of DNA synthesis by ICI 182,780 (Fig. 6). In cardiac fibroblasts, PD098059 abrogated 4-OH tamoxifen (84±11%{downarrow}; n=4) and ICI 182,780 (93±5%{downarrow}; n=4) stimulated ERK1/2 phosphorylation (Fig. 5). The 24-h exposure of cardiac fibroblasts to PD098059 significantly reduced basal [3H]thymidine uptake (70±20% {downarrow} versus basal; n=2). In this regard, the effect of PD098059 on ICI 182,780 mediated decrease of DNA synthesis was uninterpretable.


Figure 6
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  		Fig. 6 The effect of PD098059 on ICI 182,780-mediated decrease of DNA synthesis in cardiac myocytes. The 24-h exposure to PD098059 (PD;20 µM; n=4) had no effect on [3H]thymidine uptake (5±7% versus basal). The exposure to ICI 182,780 (open circles) dose-dependently decreased DNA synthesis, as compared to untreated cells (n=3). The pretreatment with PD098059 (solid circles) caused a modest non-significant attenuation of ICI 182,780 (500 nM)-mediated [3H]thymidine uptake (P=0.08; n=4), whereas an abrogation of the response was observed at higher concentrations (*denotes P<0.01 versus ICI 182,780; n=3-4 independent experiments for each concentration).

 
To confirm that the absence of an inhibitory effect of 17β-estradiol on DNA synthesis was not related at least in part via an inability to recruit ERK1/2 activation, cardiac cells were acutely exposed to the ovarian hormone. In cardiac myocytes, 1 µM 17β-estradiol increased ERK1/2 phosphorylation at 5 min (ERK1=282±22%; ERK2=263±56%; n=3; P<0.05 versus basal), and remained elevated for 30 min (Fig. 5). Likewise, 17β-estradiol (1 µM) treatment of cardiac fibroblasts increased ERK1/2 phosphorylation at 5 min (ERK1=248±19%; ERK2=283±29%; n=3; P<0.05 versus basal), and remained elevated for 30 min (Fig. 5). Lastly, the pretreatment with the MEK1/2 inhibitor PD098059 abolished 17β-estradiol-mediated ERK1/2 phosphorylation in cardiac myocytes (91±5%{downarrow}; n=3) and fibroblasts (80±10%{downarrow}; n=3).


   4. Discussion
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
References
 
Selective estrogen receptor modulators (SERMs) are a class of compounds that elicit either a partial estrogenic response, or antagonize estrogen-dependent mechanisms in a tissue selective manner [7]. The SERM tamoxifen is an effective antagonist of estrogen-dependent breast cancer, whereas tamoxifen and raloxifene mimic the beneficial action of estrogen on bone density and mineralization [7]. An alternative mode of action exclusive to SERMs is related to the identification of a novel anti-estrogen receptor not recognized by estrogen [16,17]. It is possible that this novel anti-estrogen receptor may mediate the tamoxifen induced antiproliferative action reported in several cell lines lacking estrogen receptor expression [18,19]. In cardiac cells, 17β-estradiol had a minimal effect on DNA synthesis despite estrogen receptor-{alpha}, and -β expression [14,15]. By contrast, 4-OH tamoxifen and raloxifene treatment of cardiac cells caused a dose-dependent inhibition of [3H]thymidine uptake in the absence of apoptosis. Moreover, the pretreatment of cardiac cells with 1 µM 17β-estradiol did not antagonize the action of 4-OH tamoxifen. Surprisingly, the pure estrogen receptor antagonist ICI 182,780 mimicked the SERM-dependent decrease of DNA synthesis in cardiac cells in the absence of apoptosis, and was insensitive to 17β-estradiol pretreatment. A similar antiproliferative action of ICI 182,780 was documented in an estrogen receptor-negative ovarian carcinoma cell line [19]. Thus, at least in cardiac cells, SERMs and ICI 182,780 negatively influenced DNA synthesis via a yet unidentified estrogen receptor-independent mechanism. It remains to be determined whether the inhibition of DNA synthesis in cardiac cells by SERMs and ICI 182,780 occurred via the activation of either a common or distinct receptor subtype.

In MCF-7 human mammary carcinoma cells, the estrogen receptor-independent antitumor activity of tamoxifen was associated with a decrease in the number of cells in the S-phase, and accumulation in the G0/G1 phase [23]. These latter findings of a G1-S phase blockade were in part consistent with the upregulation of the cyclin dependent kinase inhibitor p27Kip1, and the reciprocal downregulation of the DNA polymerase accessory protein proliferating cell nuclear antigen (PCNA) [24]. Indeed, the antiproliferative action of tamoxifen, acting via an estrogen receptor-independent mechanism in several distinct cell lines occurred in part via the increased expression of p27Kip1 [18,25–27]. In the present study, 4-OH tamoxifen treatment of cardiac cells increased p27Kip1 protein expression, and concomitantly downregulated PCNA levels. Analogous to 4-OH tamoxifen, the treatment of cardiac cells with ICI 182,780 increased p27Kip1 protein expression, and decreased PCNA protein levels. Likewise, the exposure of MCF-7 cells to ICI 182,780 increased p27Kip1 protein expression [28]. Thus, the disparate pattern of cell cycle protein expression represents a plausible mechanism to explain in part the decrease of DNA synthesis in cardiac cells following either 4-OH tamoxifen or ICI 182,780 treatment.

In cardiac myocytes, the acute exposure to 17β-estradiol promoted the rapid and transient activation of extracellular signal-regulated kinase (ERK1/2) [29]. Likewise, via an estrogen receptor-dependent mechanism, tamoxifen and the raloxifene analog LY117018 increased ERK activity in HeLa and vascular endothelial cells, respectively [30,31]. In the present study, 17β-estradiol and 4-OH tamoxifen treatment of cardiac cells increased ERK1/2 phosphorylation, and the detection of the estrogen receptor-{alpha} subtype on the plasma membrane supports a potential involvement [15] (and present study). However, ICI 182,780, a pure estrogen receptor antagonist with no known estrogenic properties mimicked the 4-OH tamoxifen mediated ERK1/2 phosphorylation. Based on this latter observation, the effect of ICI 182,780 on ERK1/2 phosphorylation occurred via an estrogen receptor-independent mechanism, and a similar conclusion may be relevant for 4-OH tamoxifen. Regardless the receptor implicated, 17β-estradiol, 4-OH tamoxifen and ICI 182,780 stimulated ERK1/2 phosphorylation required the recruitment of the upstream kinase MEK1/2, as the selective inhibitor PD098059 abrogated the response.

Despite the ascribed mitogenic action, an antiproliferative role of ERK has been documented. The cell cycle arrest of myoblasts and zinc mediated inhibition of colorectal cancer cell proliferation occurred via an ERK-dependent pathway [32,33]. A recent study demonstrated that the exposure of neonatal rat cardiac myocytes to atrial natriuretic peptide inhibited hypertrophy via the recruitment of ERK1/2 [22]. More importantly, ERK1/2 does not represent a requisite growth signaling event of neonatal rat cardiac myocytes and fibroblasts in response to hormonal and locally derived factors [34,35]. Thus, the hypothesis that ERK1/2 may be implicated in SERM and ICI 182,780 mediated suppression of DNA synthesis was examined. In cardiac myocytes, the MEK inhibitor PD098059 abrogated ICI 182,780 mediated decrease of DNA synthesis. In cardiac fibroblasts, PD098059 exerted a potent basal effect on [3H]thymidine uptake. Consequently, the effect of PD98059 on ICI 182,780 inhibition of DNA synthesis was uninterpretable. Nonetheless, ERK1/2 recruitment may also represent an integral antiproliferative signaling event of ICI 182,780 in cardiac fibroblasts.

Although ERK1/2 phosphorylation was observed with either 17β-estradiol or ICI 182,780 treatment, an additional and as yet unidentified pathway(s) must be required to act in concert with ERK1/2 to facilitate ICI 182,780 mediated inhibition of DNA synthesis. Second, in contrast to the present study, 17β-estradiol treatment of adult rat cardiac fibroblasts increased DNA synthesis via an ERK1/2 dependent pathway [37]. The disparate effect of 17β-estradiol on DNA synthesis observed between neonatal and adult cardiac cells may be dependent in part on the pattern of estrogen receptor subtype expression. In adult rat cardiac fibroblasts, the estrogen receptor-β was predominant subtype, whereas in neonatal rat cardiac cell, both estrogen receptor subtypes were expressed, and the estrogen receptor-{alpha} was detected on the plasma membrane [15,37] (and present study). Thus, the downstream signaling pathways coupled to each receptor subtype may be distinct and contribute in part to the disparate action of 17β-estradiol in neonatal versus adult cardiac cells. Alternatively, and as previously discussed, 17β-estradiol mediated ERK1/2 phosphorylation was not sufficient to inhibit DNA synthesis, thereby supporting the recruitment of an additional signaling event. Nonetheless, the growth-suppressing effect of ERK1/2, as observed for ICI 182,780, and atrial natriuretic peptide apparently reflects a phenotype of neonatal rat cardiac myocytes [22]. Moreover, in neonatal rat cardiac fibroblasts, recruitment of the ERK1/2 pathway did not promote growth in response to proliferative factors [34,36]. By contrast, in adult cardiac cells, ERK1/2 activation was identified as an essential signaling event of myocyte hypertrophy and fibroblast proliferation [38,39]. Consequently, the growth response of the ERK1/2 pathway was dependent on the developmental status of rat cardiac cells, and may in part explain the disparate effect of 17β-estradiol on DNA synthesis. Based on this latter observation, it is also possible that the ERK1/2-dependent inhibition of DNA synthesis in neonatal rat cardiac myocytes by ICI 182,780 and possibly SERMs may not be conserved in the adult phenotype.

In summary, this study is the first to demonstrate that DNA synthesis in neonatal rat cardiac cells were influenced by SERMs and the estrogen receptor antagonist ICI 182,780. The observed decrease of DNA synthesis in cardiac cells in response to 4-OH tamoxifen and ICI 182,780 occurred via an estrogen receptor-independent mechanism in the absence of apoptosis. The underlying mechanism(s) attributed to the action of SERMs and ICI 182,780 involved in part the disparate regulation of cell cycle proteins, and the recruitment of ERK1/2. These data provide the impetus to examine whether the estrogen receptor-independent action of SERMs and ICI 182,780 documented in neonatal cardiac cells are conserved in the adult setting, and to further elucidate their possible influence on ventricular remodeling and contractility in post-menopausal women with established cardiovascular disease.

Time for primary review 27 days.


   Acknowledgements
 
This work was supported by the Heart and Stroke Foundation of Canada and Quebec, Canadian Institutes of Health Research, and Le Fonds de Recherche de l’Institut de Cardiologie de Montréal. A. Calderone is a Chercheur-Boursier Junior II du Fonds de la recherche en santé du Québec.


   References
Top
 Abstract
 1. Introduction
 2. Methods
 3. Results
 4. Discussion
 References
 

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M. R. Meyer and M. Barton
ER{alpha}, ER{beta}, and gpER: novel aspects of oestrogen receptor signalling in atherosclerosis
Cardiovasc Res, July 6, 2009; (2009) cvp187v2.
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